Organ transplantation has become routine in many parts of the world. Transplants of liver, kidney, heart, lung and pancreas, are now regularly performed as treatment for end-stage organ disease. The outcomes of organ transplant procedures have progressively improved with the development of refinements in tissue typing, surgical techniques, and more effective immunosuppressive treatments. However, rejection of transplanted organs remains a major problem. T-lymphocytes play a central role in the immune response and they are responsible, in large measure, for the rejection of many transplanted organs. They are also responsible for the so-called graft-versus host disease in which transplanted bone marrow cells recognize and destroy MHC-mismatched host tissues. Accordingly, drugs such as cyclosporin and FK506 that suppress T-cell immunity are used to prevent transplant rejection and graft-versus-host disease. Unfortunately, these T-cell inhibiting drugs are toxic, with liver and renal toxicities limiting their use.
Autoimmune diseases encompass a whole spectrum of clinical disorders wherein a patient's immune system mistakenly attacks self, targeting the cells, tissues, and organs of the patient's own body. The following are some examples of autoimmune diseases, categorized with respect to the target organ that is principally affected by each such disease:
Nervous System:Gastrointestinal Tract:Multiple sclerosisCrohn's DiseaseMyasthenia gravisUlcerative colitisAutoimmune neuropathiesPrimary biliary cirrhosissuch as Guillain-BarréAutoimmune hepatitisAutoimmune uveitisEndocrine:Blood:Type 1 diabetes mellitusAutoimmune hemolytic anemiaAddison's DiseasePernicious anemiaGrave's DiseaseAutoimmune thrombocytopeniaHashimoto's thyroiditisVascular:Autoimmune oophoritis andTemporal arteritisorchitisAnti-phospholipid syndromeMultiple Organs and/orVasculitides such asMusculoskeletal System:Wegener's granulomatosisRheumatoid arthritisBehcet's diseaseSystemic lupus erythematosusSkin:SclerodermaPsoriasisPolymyositis, dermatomyositisDermatitis herpetiformisSpondyloarthropathies such asPemphigus vulgarisankylosing spondylitisVitiligoSjogren's syndrome
Irrespective of the particular organ(s) affected, T-lymphocytes are believed to contribute to the development of autoimmune diseases. The currently available therapies for these diseases are largely unsatisfactory and typically involve the use of glucocorticoids (e.g. methylprednisolone, prednisone), non-steroidal anti-inflammatory agents, gold salts, methotrexate, antimalarials, and other immunosuppressants such as cyclosporin and FK-506.
Thus, the search for additional immunosuppressive agents for preventing transplant rejection and for the treatment of autoimmune and inflammatory disorders occupies considerable attention in the pharmaceutical industry. Since cytokines such as interferon-gamma and tumor necrosis factor-alpha play a critical role in transplant rejection and in the pathophysiology of autoimmune disorders, much effort has been invested in the development of agents that suppress their production, secretion and/or end-organ effect.
There is an excellent track record of treating nervous and cardiovascular disorders with ion channel modulators—either openers or blockers. Ion channel blockers as a general class, represent the major therapeutic agents for treatment of stroke, epilepsy and arrhythmias. Since ion channels play a major role in the T-cell immune response, these channels may represent attractive targets for pharmaceutical immunomodulation.
The early stages of T-cell activation may be conceptually separated into pre-Ca++ and post-Ca++ events (Cahalan and Chandy 1997, Curr. Opin. Biotechnol. 8: 749). Following engagement of antigen with the T-cell antigen-receptor, activation of tyrosine kinases and the generation of inositol 1,4,5-triphosphate leads to the influx of Ca++ through store-operated calcium channels (also known as Calcium-Release Activated Calcium or CRAC channels) and the rise of cytoplasmic Ca++ concentration (Cahalan and Chandy 1997, Curr. Opin. Biotechnol. 8: 749; Kerschbaum and Cahalan 1999, Science 283: 836; Kerschbaum and Cahalan 1998; J. Gen. Physiol. 111: 521). The rise in Ca++ activates the phosphatase calcineurin, which then dephosphorylates a cytoplasmically localized transcription factor (N-FAT) enabling it to accumulate in the nucleus and bind to a promoter element of the interleukin-2 gene. Along with parallel events involving the activation of protein kinase C and ras, gene transcription leads to lymphokine secretion and to lymphocyte proliferation. Some genes require long-lasting Ca++ signals while others require only a transient rise of Ca++. Furthermore, Ca++ immobilization of the T-cell at the site of antigen presentation helps to cement the interaction between T-cell and the antigen-presenting cell and thereby facilitate local signaling between the cells (Negulescu 1996, Immunity 4:421).
Ion channels underlie the Ca++ signal of T-lymphocytes. Ca++ ions move across the plasma membrane through a channel termed the store-operated Ca++ channel or the CRAC channel which is activated by depletion of internal calcium stores like the endoplasmic reticulum (Cahalan and Chandy 1997, Curr. Opin. Biotechnol. 8: 749). Two distinct types of potassium channels indirectly determine the driving force of calcium entry through the store-operated Ca2+ channel (Cahalan and Chandy 1997, Curr. Opin. Biotechnol. 8: 749). The first is the voltage-gated Kv1.3 channel (Cahalan 1985, J. Physiol. 385: 197; Grissmer 1990, Proc. Natl. Acad. Sci. USA 87: 9411; Verheugen 1995, J. Gen. Physiol. 105: 765; Aiyar 1996, J. Biol. Chem. 271: 31013; Cahalan and Chandy 1997, Curr. Opin. Biotechnol. 8: 749) and the second is the intermediate-conductance calcium-activated potassium channel, IKCa1 (Grissmer 1993, J. Gen. Physiol. 102: 601; Fanger 1999 J. Biol. Chem. 274: 5746; Rauer 1999, J. Biol. Chem. 274: 21885) which is also known as IK1 (VanDorpe 1998, J. Biol. Chem. 273: 21542), hSK4 (Joiner 1997, Proc. Natl. Acad. Sci. USA 94: 11013; Khanna 1999, J. Biol. Chem. 274: 14838) and hKCa4 (Lodgson 1997, J. Biol. Chem. 272: 32723; Ghanshani 1998, Genomics 51: 160). When these potassium channels open, the resulting efflux of K+ hyperpolarizes the membrane, which in turn accentuates the entry of Ca++, which is absolutely required for downstream activation events (Cahalan and Chandy 1997, Curr. Opin. Biotechnol. 8: 749). Blockers of the Kv1.3 and IKCa1 channels suppress human T-cell activation, when applied independently, and produce greater suppression when applied together (DeCoursey 1984, Nature 307: 465; Chandy J. Exp. Med. 160: 369; Koo 1997, J. Immunol. 158: 5120; Nguyen 1995, Mol. Pharmacol. 50: 1672; Hanson 1999, Br. J. Pharmacol. 126:1707; Kalman 1998, J. Biol. Chem. 278: 32697; Khanna 1999, J. Biol. Chem. 274: 14838; Jensen 1999; Proc. Natl. Acad. Sci. USA 96: 10917). One mechanism for the immunosuppression by K+ channel blockers is via membrane depolarization, which reduces Ca++ entry through CRAC channels in the T-cell membrane, which in turn leads to suppression of calcium-dependent signaling events during human T-cell activation (Cahalan and Chandy 1997, Curr. Opin. Biotechnol. 8: 749; Koo 1999, Cell. Immunol. 197: 99).
Clotrimazole, a non-selective inhibitor of IKCa1, suppresses mitogen-stimulated T-cell activation, especially of pre-activated cells (Khanna 1999, J. Biol. Chem. 274: 14838; Jensen 1999, Proc. Natl. Acad. Sci. USA 96: 10917). Clotrimazole and related imidazoles, other than the compounds of this invention, have also previously been described for use in treating rheumatoid arthritis, an autoimmune disorder (Wojtulewski 1980, Ann. Rheum. Dis. 39: 469; Wyburn-Mason 1976, U.S. Pat. No. 4,073,922; Wyburn-Mason 1987, U.S. Pat. No. 183,941; Wyburn-Mason 1979, U.S. Pat. No. 4,218,449). However, clotrimazole shows considerable toxicity with increasing doses, toxicity being primarily associated with its potent (nanomolar) inhibition of cytochrome P450 enzymes (Wojtulewski 1980, Ann. Rheum. Dis. 39: 469; Burgess 1972 Antimicrob. Agents Chemother. 2: 423; Brugnara 1996, J. Clin Invest 97: 1227). Thus, there clearly is a need for newer analogs that block IKCa1 without concomitant inhibition of cytochrome P450-dependent enzymes.
Other patents have described the use of clotrimazole, related azole antimycotics (e.g., miconazole and econazole) and related aromatic halides for the treatment of cancer (Halperin 1994, WO 96/08240; Halperin 1997 U.S. Pat. No. 5,633,274), but only at micromolar concentrations (Benzaquen 1995, Nat. Med. 1: 534), substantially greater than the concentrations required to block the IKCa1 channel (˜20–100 nM), suggesting that the mechanism of suppression of proliferation might be unrelated to channel block. Also at micromolar concentrations, clotrimazole, related azole antimycotics (e.g., miconazole and econazole) and related aromatic halides have been described for use in the treatment of arteriosclerosis as a hyperproliferative disease (Halperin 1994, WO 94/189680 and U.S. Pat. No. 5,358,959), and for the treatment of diseases characterized by neovascularization (Halperin 1996, U.S. Pat. No. 5,512,591; Halperin 1997, U.S. Pat. No. 5,643,936 and U.S. Pat. No. 5,591,763).
At least some of the triarylmethyl-1H-pyrazole compounds of the present invention have also previously been described in PCT International Publication WO 97/34599 entitled USE OF CLOTRIMAZOLE AND RELATED COMPOUNDS IN THE TREATMENT OF DIARRHEA, as being useable for the treatment of diarrhea, although they do not constitute preferred embodiments of the inventions.
Also, PCT International Publication WO/97/34589 entitled TRIARYL METHANE COMPOUNDS FOR SICKLE CELL DISEASE describes various substituted triarylmethane compounds as effective treatments for sickle cell disease due to their ability to inhibit ion flux through the calcium activated potassium channel (Gardos channel) of erythrocytes, which has now been shown to be encoded by the IKCa1 gene (VanDorpe 1998, J. Biol. Chem. 273: 21542). Clotrimazole, the preferred compound in this invention is in phase II trials for the treatment of sickle cell disease gene (VanDorpe 1998, J. Biol. Chem. 273: 21542), but at higher doses causes toxic side effects most likely due to its inhibition of cytochrome P450 enzymes. The PCT International Publication WO/97/34589 also describes various substituted triarylmethane compounds as effective treatments for diseases characterized by unwanted or abnormal cell proliferation (the examples cited being melanoma cells and fibroblast proliferation), but at only micromolar concentrations (Benzaquen 1995, Nat. Med. 1: 534; Halperin 1997 U.S. Pat. No. 5,633,274; PCT application WO97/34589; PCT application WO/97/08240) which are substantially higher than that required for block of the IKCa1 channel (half-block at 20–100 nM), suggesting that the mechanism of suppression of cell proliferation might be unrelated to channel block. Furthermore, since the three compounds used to support this claim, clotrimazole, ketoconazole and miconazole (Benzaquen 1995, Nat. Med. 1: 534) also inhibit cytochrome P450 enzymes at nanomolar concentrations (Mason 1987, Steroids 50: 179; Morris 1992, FASEB J. 6: 752), the mechanism of suppression of abnormal proliferation may be related to inhibition of these enzymes. Another possible mechanism for suppression of proliferation stated in PCT application WO/97/34589 is non-specific cytotoxicity. Therefore, the claims in PCT application WO/97/34589 that suppression of abnormal proliferation is due solely to alteration of transmembrane ion fluxes cannot be substantiated.
WO 97/34589 does not describe or suggest that the substituted triarylmethane compounds disclosed therein are capable of selectively blocking the calcium activated potassium channels encoded by the IKCa1 gene in resting and activated T-lymphocytes, or that such compounds would, alone or in combination with other inhibitors of T-cell signaling cascades, suppress antigen-, cytokine- and/or mitogen-stimulated calcium-entry through store-operated calcium channels, and/or cytokine production and/or activation of human T-lymphocytes, without concomitant inhibition of cytochrome P450 enzymes, leading to immunosuppressive activity when administered to mammalian patients.
Given the shortcomings associated with the currently available modes of therapy for autoimmune disorders, transplant rejection and graft-versus-host disease, there remains a need for the development of new immunosuppressive drugs that are capable of selectively inhibiting the activation of lymphocytes with minimal side effects, and without affecting the cytochrome P450 enzyme system.